Primer combination for identifying genetic sex of sturgeon, and kit and use thereof

Abstract

Provided are a primer combination for identifying the genetic sex of sturgeon, and a kit and a use thereof. By means of two pairs of primers, the genetic sex of sturgeon can be identified simply and quickly, and experimental errors can be effectively avoided, thereby improving the accuracy of sex identification for sturgeon. Moreover, the present invention is not limited by the age of sturgeon, will not cause significant damage to sturgeon, and further has good universality. The present invention can help with sex controlled breeding and germplasm resource protection and utilization of sturgeon and provide technical support for biological diversity research and Yangtze River protection work.

Description

A primer combination, kit and application for identifying the genetic sex of sturgeon

[0001] The present application claims priority to the Chinese patent application No. 2024108156670 filed on June 21, 2024, and entitled "A primer combination, kit and application for identifying the genetic sex of sturgeon", the content of which is incorporated herein by reference in its entirety. TECHNICAL FIELD

[0002] The present application relates to molecular biological technology, and in particular to a primer combination, kit and application for identifying the genetic sex of sturgeon. BACKGROUND

[0003] Sturgeon is the abbreviation of Acipenseriformes, which is the only large cartilaginous bony fish in the class of bony fish. Sturgeon matures at an old age, spawns in rivers, and most species have the habit of anadromous migration. There are 27 existing species in the world, which have great academic significance and economic value. However, the intensification of human activities and overfishing in the past century have led to a sharp decline in the resources of all sturgeon species, and all species are currently in different degrees of endangered state, which has attracted widespread attention from the society.

[0004] Sturgeon has no obvious external secondary sex characteristics such as pearl stars, so it is difficult to distinguish male sturgeon from immature female sturgeon in non-spawning areas. In the spawning area, sperm can generally be squeezed out of the genital pore of mature male sturgeon, while female sturgeon generally have an enlarged abdomen and a thicker waist, but small individual female sturgeon that will spawn for the first time are often confused with male sturgeon without sperm and immature female sturgeon. According to the external morphology of the genital pore of sturgeon, the male and female can be distinguished. By observing the wild sturgeon of Acipenser oxyrhynchus, Acipenser brevirostrum, Acipenser oxyrhynchus and Acipenser oxyrhynchus, it is found that the female genital pore is generally in the shape of "O", while the male genital pore is generally in the shape of "Y". The accuracy of this identification method reaches 82% in live specimens, but only 29% in dead specimens. Some scholars have measured the head quantitative traits to identify the sex of Russian sturgeon, with an accuracy of more than 90%. For sturgeons whose gonads have developed to the second stage or above, the most direct and convenient method of sex identification is to distinguish the ovary and testis through laparoscopic surgery. However, the above-mentioned methods of sex identification of sturgeon can only identify sturgeons that have developed to a certain age, and some methods of sex identification of sturgeon can cause great damage to sturgeon, such as laparoscopic surgery. There are reports about the sex identification method of sturgeon female-specific DNA fragments. In the operation of this method, if DNA extraction is unsuccessful, the wrong or missing addition of drugs occurs, the sturgeon to be sex identified will be misjudged as male sturgeon, which will cause errors in the sex identification of sturgeon.

[0005] Exploring the regulatory mechanisms of sturgeon sex differentiation and establishing new technologies for breeding all-female or high-proportion female sturgeon is of significant theoretical and practical importance for the protection and development of its germplasm resources. The development of sex-specific molecular markers can help accurately and rapidly obtain phenotypes and determine sex, thereby facilitating artificial breeding and selection. It also contributes to research on sex-controlled breeding technologies in commercial production, ultimately leading to the establishment of a monosex system. Therefore, developing a molecular marker for accurately identifying sturgeon sex has immense practical value in the artificial breeding of sturgeon.

[0006] Summary of the Invention

[0007] This application provides a primer combination, reagent kit, and application for identifying the genetic sex of sturgeon, which improves the accuracy of sex identification in sturgeon and allows for sex identification throughout the entire life cycle of sturgeon.

[0008] This application also provides a method for identifying the genetic sex of sturgeon, which uses the above-mentioned primer combination or kit. The method is simple to operate, produces accurate results, and can be used to identify the sex of sturgeon throughout its entire life cycle.

[0009] This application provides a primer pair for identifying the genetic sex of sturgeon, including a first primer pair and a second primer pair; the first primer pair includes a first primer and a second primer, the nucleotide sequence of the first primer is shown in SEQ ID NO:1, and the nucleotide sequence of the second primer is shown in SEQ ID NO:2; the second primer pair includes a third primer and a fourth primer, the nucleotide sequence of the third primer is shown in SEQ ID NO:3, and the nucleotide sequence of the fourth primer is shown in SEQ ID NO:4.

[0010] The primer combination described above includes a first primer pair for amplifying a female sturgeon-specific DNA fragment, the nucleotide sequence of which is shown in SEQ ID NO:5, and the fragment size is 473 bp; and a second primer pair for amplifying a DNA fragment shared by both female and male sturgeon, the nucleotide sequence of which is shown in SEQ ID NO:6, and the fragment size is 717 bp.

[0011] The primer combination described above includes at least one of the following: Chinese sturgeon, Yangtze sturgeon, small sturgeon, Shire sturgeon, large mixed sturgeon, Siberian hybrid sturgeon, Siberian sturgeon, Kaluga sturgeon, Russian sturgeon, shin sturgeon, European sturgeon, lake sturgeon, naked sturgeon, Persian sturgeon, high-headed sturgeon, and shovelnose sturgeon.

[0012] This application provides a kit for genetic sex identification of sturgeon, comprising the primer combination described above.

[0013] The kit as described above, wherein the kit further comprises at least one of water, PCR buffer, dNTPs, DNA polymerase, loading buffer.

[0014] The application provides an application of the primer combination or the kit in identifying the genetic sex of the sturgeon.

[0015] The application further provides a method for identifying the genetic sex of the sturgeon, comprising the following steps:

[0016] extracting DNA of a sturgeon sample to be detected;

[0017] using the primer combination or the kit to perform PCR amplification on the DNA of the sturgeon sample to be detected as a template, so as to obtain a PCR amplification product;

[0018] performing agarose gel electrophoresis detection on the PCR amplification product, when two bands of 473 bp and 717 bp appear in the detection result, it is identified that the genetic sex of the sturgeon is female; and when one band of 717 bp appears in the detection result, it is identified that the genetic sex of the sturgeon is male.

[0019] The method as described above, wherein the sample of the sturgeon to be detected comprises at least one of a fin strip and mucus of the sturgeon.

[0020] The method as described above, wherein in the PCR amplification, the PCR reaction system comprises 3-5 μL of the DNA template, 0.5-1.5 μL of the first primer, 0.5-1.5 μL of the second primer, 0.25-0.75 μL of the third primer, 0.25-0.75 μL of the fourth primer, 10-15 μL of 2x Taq Master Mix and 4-6 μL of ddH2O.

[0021] The method as described above, wherein in the PCR amplification, the PCR amplification procedure comprises 93-95℃ pre-denaturation for 3 min; 93-95℃ denaturation for 10 s, 54-56℃ recombination for 10 s, 72℃ extension for 19 s, a total of 35 cycles; and finally 72℃ extension for 10 min.

[0022] The primer combination provided by the application provides two pairs of primers, which can avoid experimental errors caused by unsuccessful DNA extraction, wrong or missing addition of medicines, thereby improving the accuracy of the sturgeon sex identification. Meanwhile, the primer combination provided by the application is not limited by the age of the sturgeon, and can be used for sex identification in the whole life cycle of the sturgeon. The application provides help for sturgeon sex control breeding and utilization of germplasm resources, and provides technical support for biodiversity research and Yangtze River protection. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 is an electropherogram of genetic sex identification of Acipenser sinensis in Example 1;

[0024] Figure 2 is an electropherogram of genetic sex identification of Acipenser dabryanus in Example 2;

[0025] Figure 3 is an electropherogram of genetic sex identification of Acipenser ruthenus in Example 3;

[0026] Figure 4 is an electropherogram of genetic sex identification of Acipenser schrencki in Example 4;

[0027] Figure 5 is an electropherogram of genetic sex identification of Acipenser schrencki ×Huso dauricus♀ in Example 5;

[0028] Figure 6 is an electropherogram of genetic sex identification of Acipenser schrenckii ×Acipenser baerii♀ in Example 6;

[0029] Figure 7 is an electropherogram of genetic sex identification of Acipenser baerii in Example 7;

[0030] Figure 8 is an electropherogram of genetic sex identification of Huso dauricus in Example 8;

[0031] Figure 9 is an electropherogram of genetic sex identification of Acipenser gueldenstaedti in Example 9;

[0032] Figure 10 is an electropherogram of genetic sex identification of Acipenser stellatus in Example 10;

[0033] Figure 11 is an electropherogram of genetic sex identification of Huso huso in Example 11;

[0034] Figure 12 is an electropherogram of genetic sex identification of Acipenser fulvescens in Example 12;

[0035] Figure 13 is an electropherogram of genetic sex identification of Acipenser nudiventris in Example 13;

[0036] Figure 14 is an electropherogram of genetic sex identification of Acipenser persicus in Example 14;

[0037] Figure 15 is an electropherogram of genetic sex identification of Acipenser transmontanus in Example 15;

[0038] Figure 16 is an electropherogram of genetic sex identification of Acipenser medirostris in Example 16. DETAILED DESCRIPTION

[0039] In order for those skilled in the art to better understand the scheme of the present application, the present application is further described in detail below. The following specific embodiments are only used to describe the principles and characteristics of the present application, and the examples are used to explain the present application, but not to limit the scope of the present application. Based on the embodiments of the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0040] The first aspect of the present application provides a primer combination for identifying the genetic sex of sturgeon, comprising a first primer pair and a second primer pair; the first primer pair comprises a first primer and a second primer, the nucleotide sequence of the first primer is shown in SEQ ID NO: 1, and the nucleotide sequence of the second primer is shown in SEQ ID NO: 2; the second primer pair comprises a third primer and a fourth primer, the nucleotide sequence of the third primer is shown in SEQ ID NO: 3, and the nucleotide sequence of the fourth primer is shown in SEQ ID NO: 4.

[0041] The primer combination for identifying the genetic sex of sturgeon comprises a first primer pair and a second primer pair, in the first primer pair of the present application, the first primer is an upstream primer, and the second primer is a downstream primer; in the second primer pair of the present application, the third primer is an upstream primer, and the fourth primer is a downstream primer.

[0042] The present application obtains a primer combination for identifying the genetic sex of sturgeon according to sturgeon genomic DNA. When designing the primer combination, the present application not only considers how to simply and quickly identify the genetic sex of sturgeon, but also takes how to avoid experimental errors as a core factor, thereby obtaining a primer combination comprising a first primer pair and a second primer pair.

[0043] Among them, the first primer pair is used to simply and quickly identify whether the genetic sex of sturgeon is female, and the second primer pair is used to avoid experimental errors caused by unsuccessful DNA extraction, wrong or missing addition of drugs. When the second primer pair amplifies a DNA fragment common to female sturgeons and male sturgeons, it proves that the experimental operation is correct or the experimental materials are normal, and there is no situation such as unsuccessful DNA extraction, wrong or missing addition of drugs. At this time, the accuracy of the result amplified by the first primer pair is higher.

[0044] In addition, in order to reduce the influence of serious DNA degradation leading to DNA fragment loss on the identification of the genetic sex of sturgeon, a primer pair as shown in SEQ ID NO: 1-SEQ ID NO: 4 is designed, and the amplification fragments obtained based on the primer pair are not only located on the same chromosome, but also the distance between the two is not greater than 20kbp. Therefore, even if serious DNA degradation occurs, it will not affect the identification result.

[0045] Therefore, the primer combination of the present application not only has the ability to simply and quickly identify the genetic sex of sturgeon, but also avoids experimental errors, thereby improving the accuracy of sturgeon sex identification.

[0046] Further, the first primer pair is used to amplify a female sturgeon specific DNA fragment, the nucleotide sequence of the female sturgeon specific DNA fragment is shown in SEQ ID NO: 5, and the fragment size is 473bp. The second primer pair is used to amplify a female sturgeon and male sturgeon common DNA fragment, the nucleotide sequence of the female sturgeon and male sturgeon common DNA fragment is shown in SEQ ID NO: 6, and the fragment size is 717bp.

[0047] In order to ensure the accuracy of the amplification result, the present application limits the amplified fragments. When the amplified result does not meet the above-mentioned limitation, it means that the experimental operation is wrong or the experimental material may have a problem. Therefore, the accuracy of the result can be ensured by re-experimenting.

[0048] The present application does not limit that the primer combination can be used for the genetic sex limitation of various sturgeons, for example, it can be Chinese sturgeon, Yangtze sturgeon, small body sturgeon, Schizothorax chongmi, big hybrid sturgeon, Siberian hybrid sturgeon, Siberian sturgeon, Huso dauricus, Russian sturgeon, Acipenser sturio, Acipenser gueldenstaedtii, Acipenser ruthenus, Acipenser sturio, Acipenser persicus, Acipenser oxyrhynchus, Acipenser sturio.

[0049] Among them, the big hybrid sturgeon refers to the hybrid F1 generation of Schizothorax chongmi and Huso dauricus. In an embodiment of the present application, the big hybrid sturgeon used is the hybrid F1 generation with Schizothorax chongmi as the father and Huso dauricus as the mother. The Siberian hybrid sturgeon is the hybrid F1 generation of Siberian sturgeon and Schizothorax chongmi. In an embodiment of the present application, the Siberian hybrid sturgeon used is the hybrid F1 generation with Schizothorax chongmi as the father and Siberian sturgeon as the mother.

[0050] The second aspect of the present application provides a kit for identifying the genetic sex of sturgeon, comprising the primer combination provided in the first aspect of the present application.

[0051] Since the kit comprises the primer combination provided in the first aspect of the present application, the kit also has the ability to simply and quickly identify the genetic sex of sturgeon, and avoids experimental errors, thereby improving the accuracy of sturgeon sex identification.

[0052] In a specific embodiment, the kit further comprises at least one of water, PCR buffer, dNTPs, DNA polymerase, loading buffer.

[0053] The water can provide the ion environment and water molecules required for the PCR reaction, so that the interaction between various molecules in the reaction can proceed smoothly, and can also help to maintain the temperature stability of the reaction system, ensuring the reliability and accuracy of the reaction. The PCR buffer can adjust the pH value of the reaction system, so that the action environment of the Taq DNA polymerase is maintained to be alkaline. The Mg 2+ The dNTPs are four free deoxyribonucleoside triphosphates, which are essential raw materials for PCR amplification. The DNA polymerase is a kind of enzyme that catalyzes the polymerization of substrate dNTPs molecules to form daughter DNA with parent DNA as template. The DNA polymerase in PCR technology generally also has thermal stability. The loading buffer can increase the sample density to prevent the sample from floating out of the sample hole. The indicator contained in the loading buffer can also show the progress of electrophoresis. Some loading buffers also contain sodium dodecyl sulfate, which can promote the denaturation of DNA polymerase and avoid the residual DNA polymerase from binding to the DNA double strand to affect the electrophoretic migration rate.

[0054] The third aspect of the present application provides an application of the primer combination or the kit in identifying the genetic sex of sturgeon.

[0055] In a specific embodiment, Figure 1 is an electrophoresis detection result of the genetic sex identification of Acipenser sinensis provided by an embodiment of the present application. As shown in Figure 1, when two bands of 473 bp and 717 bp appear in the detection result, the genetic sex of the sturgeon is female; when a band of 717 bp appears in the detection result, the genetic sex of the sturgeon is male.

[0056] The fourth aspect of the present application provides a method for identifying the genetic sex of sturgeon, comprising the following steps:

[0057] Extracting the DNA of the sturgeon sample to be detected;

[0058] Using the DNA of the sturgeon sample to be detected as a template, using the primer combination or the kit for PCR amplification to obtain a PCR amplification product;

[0059] Performing agarose gel electrophoresis detection on the PCR amplification product. When two bands of 473 bp and 717 bp appear in the detection result, the genetic sex of the sturgeon is identified to be female; when a band of 717 bp appears in the detection result, the genetic sex of the sturgeon is identified to be male.

[0060] According to the primer combination provided in the first aspect of the present application, the DNA of the sturgeon sample to be detected is subjected to PCR detection and agarose gel electrophoresis detection, and then the genetic sex of the sturgeon sample to be detected can be identified according to the number and size of the bands displayed in the detection, i.e., the sex of the sturgeon sample to be detected is identified as female or male. The identification method provided in the present application only needs to extract a small amount of DNA from the sturgeon to be detected, and will not cause significant impact and damage to the sturgeon, nor will it be limited by the age of the sturgeon. The genetic sex of the sturgeon can be identified throughout its life cycle.

[0061] In detail, when the identification is performed according to the above steps, the sample of the sturgeon to be detected includes at least one of a fin strip of the sturgeon and mucus, and the mucus can be the mucus on the surface of the sturgeon. The genomic DNA of the sample of the sturgeon to be detected can be extracted by using conventional technical means in the art, for example, the CTAB method can be used.

[0062] Further, in the PCR amplification, the PCR reaction system includes 3-5 μL of DNA template, 0.5-1.5 μL of the first primer, 0.5-1.5 μL of the second primer, 0.25-0.75 μL of the third primer, 0.25-0.75 μL of the fourth primer, 10-15 μL of 2×Taq Master Mix, and 4-6 μL of ddH2O.

[0063] In addition, when the PCR amplification program includes 93-95℃ pre-denaturation for 3 min; 93-95℃ denaturation for 10 s, 54-56℃ annealing for 10 s, 72℃ extension for 19 s, a total of 35 cycles; and finally 72℃ extension for 10 min, it helps to improve the specificity of the PCR reaction, and further improves the accuracy of the identification of the genetic sex of the sturgeon.

[0064] Finally, the PCR amplification product is detected according to the conventional agarose gel electrophoresis detection method in the art, and the genetic sex of the sturgeon sample to be detected can be identified according to the number and size of the bands displayed in the agarose gel electrophoresis.

[0065] In the following, the technical solutions of the present application will be further explained and described in combination with specific examples.

[0066] In the following examples, DL5000 DNA molecular weight marker (DNA Marker, abbreviated as M) is used for agarose gel electrophoresis, and the electrophoresis bands from top to bottom are 5000 bp, 3000 bp, 2000 bp, 1000 bp, 750 bp, 500 bp, 250 bp and 100 bp. The experimental methods not specifically mentioned in the following examples are usually performed according to the conventional conditions or the conditions recommended by the manufacturers. The reagents used are commercially available or can be obtained from public channels, unless otherwise specified.

[0067] The primer sequences involved in the following examples are shown in Table 1:

[0068] Table 1

[0069] Example 1: Identification of genetic sex of Acipenser sinensis

[0070] After the genetic sex of Acipenser sinensis was identified by spawning and milking, fin rays of 12 female Acipenser sinensis and 12 male Acipenser sinensis were taken, wherein the Acipenser sinensis were from the Acipenser sinensis Research Institute of China Three Gorges Corporation. DNA of the above-mentioned 24 Acipenser sinensis was extracted from the fin rays of the Acipenser sinensis using a DNA extraction kit to prepare DNA samples. The integrity of the DNA samples was identified by 1.5% agarose gel electrophoresis, and the OD value of the DNA samples was determined using a UV spectrophotometer and then diluted to 50 ng / μL.

[0071] According to the female Acipenser sturio specific DNA fragment, primers XYJD15F and XYJD15R were designed, wherein the nucleotide sequence of the female Acipenser sturio specific DNA fragment is shown in SEQ ID NO: 5, and the fragment size is 473 bp. According to the female Acipenser sturio and male Acipenser sturio common DNA fragment, primers XYXY38F and XYXY38R were designed, wherein the nucleotide sequence of the female Acipenser sturio and male Acipenser sturio common DNA fragment is shown in SEQ ID NO: 6, and the fragment size is 717 bp.

[0072] The above-mentioned DNA samples were used as templates, and PCR technology was used for amplification to obtain PCR products. The PCR amplification system was 25 μL, including 4 μL of DNA template, 1 μL of XYJD15F, 1 μL of XYJD15R, 0.5 μL of XYXY38F, 0.5 μL of XYXY38R, 12.5 μL of 2×Taq Master Mix (With Dye), and 5.5 μL of ddH2O. The PCR amplification program included 94°C pre-denaturation for 3 min, 94°C denaturation for 10 s, 56°C annealing for 10 s, 72°C extension for 19 s, a total of 35 cycles, and finally 72°C extension for 10 min and 4°C storage. The PCR products were added to 3% agarose gel, the voltage was set to 120 V, and the time was set to 25 min for electrophoresis, and a gel imaging system was used to analyze the agarose gel after electrophoresis.

[0073] As shown in FIG. 1, it can be seen that 12 female (Female) Acipenser sinensis all appeared two bands with sizes of 473 bp and 717 bp, and 12 male (Male) Acipenser sinensis all appeared one band with a size of 717 bp. The experimental results are consistent with the identification results of the genetic sex of Acipenser sinensis obtained by spawning and milking.

[0074] Example 2: Identification of genetic sex of Acipenser dabryanus

[0075] This example 2 can refer to the steps of example 1, the difference is that after determining the genetic sex of the Yangtze sturgeon by spawning and milking, the fin rays of 12 female Yangtze sturgeons and 12 male Yangtze sturgeons are taken, wherein the Yangtze sturgeons come from the Chinese Sturgeon Institute of China Three Gorges Corporation.

[0076] As shown in Figure 2, it can be known that 12 female Yangtze sturgeons all appear two bands, the sizes are 473bp and 717bp respectively; 12 male Yangtze sturgeons all appear one band, the size is 717bp. The experimental results are consistent with the identification results of the genetic sex of the Yangtze sturgeon obtained by spawning and milking.

[0077] Example 3: Identification of the genetic sex of the Pseudobrama

[0078] This example 3 can refer to the steps of example 1, the difference is that after determining the genetic sex of the Pseudobrama by dissection, the fin rays of 12 female Pseudobrama and 12 male Pseudobrama are taken, wherein the Pseudobrama comes from the Hubei Yichang Sturgeon Farm in China.

[0079] As shown in Figure 3, it can be known that 12 female Pseudobrama all appear two bands, the sizes are 473bp and 717bp respectively; 12 male Pseudobrama all appear one band, the size is 717bp. The experimental results are consistent with the identification results of the genetic sex of the Pseudobrama obtained by dissection.

[0080] Example 4: Identification of the genetic sex of the Polyodon spathula

[0081] This example 4 can refer to the steps of example 1, the difference is that after determining the genetic sex of the Polyodon spathula by dissection, the fin rays of 12 female Polyodon spathula and 12 male Polyodon spathula are taken, wherein the Polyodon spathula comes from the Hubei Yichang Sturgeon Farm in China.

[0082] As shown in Figure 4, it can be known that 12 female Polyodon spathula all appear two bands, the sizes are 473bp and 717bp respectively; 12 male Polyodon spathula all appear one band, the size is 717bp. The experimental results are consistent with the identification results of the genetic sex of the Polyodon spathula obtained by dissection.

[0083] Example 5: Identification of the genetic sex of the big hybrid sturgeon

[0084] This example 5 can refer to the steps of example 1, the difference is that the big hybrid sturgeon used in this experiment is the hybrid offspring of the male parent Polyodon spathula and the female parent Pseudobrama, after determining the genetic sex of the big hybrid sturgeon by dissection, the fin rays of 12 female big hybrid sturgeons and 12 male big hybrid sturgeons are taken, wherein the big hybrid sturgeon comes from the Hubei Yichang Sturgeon Farm in China.

[0085] As shown in Figure 5, it can be known that 12 female big hybrid sturgeon all appear two bands, and the sizes are 473bp and 717bp respectively; 12 male big hybrid sturgeon all appear one band, and the size is 717bp. The experimental results are consistent with the genetic sex identification results of big hybrid sturgeon obtained by dissection.

[0086] Example 6: Genetic sex identification of Siberian hybrid sturgeon

[0087] The steps of this example 6 can refer to the steps of example 1, and the difference is that the Siberian hybrid sturgeon used in this experiment is the hybrid offspring of female Siberian sturgeon and male Acipenser schrenckii. After the genetic sex of Siberian hybrid sturgeon is determined by dissection, the fin rays of 12 female Siberian hybrid sturgeons and 12 male Siberian hybrid sturgeons are taken, wherein the Siberian hybrid sturgeon comes from the sturgeon breeding farm in Yichang, Hubei, China.

[0088] As shown in Figure 6, it can be known that 12 female Siberian hybrid sturgeon all appear two bands, and the sizes are 473bp and 717bp respectively; 12 male Siberian hybrid sturgeon all appear one band, and the size is 717bp. The experimental results are consistent with the genetic sex identification results of Siberian hybrid sturgeon obtained by dissection.

[0089] Example 7: Genetic sex identification of Siberian sturgeon

[0090] The steps of this example 7 can refer to the steps of example 1, and the difference is that after the genetic sex of Siberian sturgeon is determined by dissection, the fin rays of 12 female Siberian sturgeons and 12 male Siberian sturgeons are taken, wherein the Siberian sturgeon comes from the sturgeon breeding farm in Yichang, Hubei, China.

[0091] As shown in Figure 7, it can be known that 12 female Siberian sturgeon all appear two bands, and the sizes are 473bp and 717bp respectively; 12 male Siberian sturgeon all appear one band, and the size is 717bp. The experimental results are consistent with the genetic sex identification results of Siberian sturgeon obtained by dissection.

[0092] Example 8: Genetic sex identification of Acipenser dabryanus

[0093] The steps of this example 8 can refer to the steps of example 1, and the difference is that after the genetic sex of Acipenser dabryanus is determined by dissection, the fin rays of 12 female Acipenser dabryanus and 12 male Acipenser dabryanus are taken, wherein the Acipenser dabryanus comes from the sturgeon breeding farm in Beijing, China.

[0094] As shown in Figure 8, it can be known that 12 female Acipenser dabryanus all appear two bands, and the sizes are 473bp and 717bp respectively; 12 male Acipenser dabryanus all appear one band, and the size is 717bp. The experimental results are consistent with the genetic sex identification results of Acipenser dabryanus obtained by dissection.

[0095] Example 9: Identification of genetic sex of Russian sturgeon

[0096] This example 9 can refer to the steps of example 1, the difference is that after determining the genetic sex of Russian sturgeon by dissection, the fin rays of 12 female Russian sturgeons and 12 male Russian sturgeons are taken, wherein the Russian sturgeons come from Beijing Sturgeon Farm in China.

[0097] As shown in Figure 9, it can be seen that 12 female Russian sturgeons all appear two bands, the sizes are 473bp and 717bp respectively; 12 male Russian sturgeons all appear one band, the size is 717bp. The experimental results are consistent with the identification results of genetic sex of Russian sturgeon obtained by dissection.

[0098] Example 10: Identification of genetic sex of Polyodon spathula

[0099] This example 10 can refer to the steps of example 1, the difference is that after determining the genetic sex of Polyodon spathula by dissection, the fin rays of 12 female Polyodon spathulas and 12 male Polyodon spathulas are taken, wherein the Polyodon spathulas come from Beijing Sturgeon Farm in China.

[0100] As shown in Figure 10, it can be seen that 12 female Polyodon spathulas all appear two bands, the sizes are 473bp and 717bp respectively; 12 male Polyodon spathulas all appear one band, the size is 717bp. The experimental results are consistent with the identification results of genetic sex of Polyodon spathula obtained by dissection.

[0101] Example 11: Identification of genetic sex of Acipenser sturio

[0102] This example 11 can refer to the steps of example 1, the difference is that after determining the genetic sex of Acipenser sturio by endoscopy, the fin rays of 12 female Acipenser sturio and 12 male Acipenser sturio are taken, wherein the Acipenser sturio comes from Zhejiang Sturgeon Farm in China.

[0103] As shown in Figure 11, it can be seen that 12 female Acipenser sturio all appear two bands, the sizes are 473bp and 717bp respectively; 12 male Acipenser sturio all appear one band, the size is 717bp. The experimental results are consistent with the identification results of genetic sex of Acipenser sturio obtained by endoscopy.

[0104] Example 12: Identification of genetic sex of Acipenser brevirostrum

[0105] This example 12 can refer to the steps of example 1, the difference is that after determining the genetic sex of Acipenser brevirostrum by dissection, the fin rays of 12 female Acipenser brevirostrum and 12 male Acipenser brevirostrum are taken, wherein the Acipenser brevirostrum comes from Xinjiang Sturgeon Farm in China.

[0106] As shown in Figure 12, it can be known that 12 female lake sturge appear two bands, and the sizes are 473bp and 717bp respectively; 12 male lake sturge appear one band, and the size is 717bp. The experimental results are consistent with the identification results of genetic sex of lake sturge obtained by dissection.

[0107] Example 13: Identification of genetic sex of naked sturgeon

[0108] The steps of this example 13 can refer to the steps of example 1, and the difference is that after the genetic sex of naked sturgeon is determined by dissection, the fin rays of 12 female naked sturgeons and 12 male naked sturgeons are taken, wherein the naked sturgeons come from the sturgeon breeding farm in Xinjiang, China.

[0109] As shown in Figure 13, it can be known that 12 female naked sturgeons appear two bands, and the sizes are 473bp and 717bp respectively; 12 male naked sturgeons appear one band, and the size is 717bp. The experimental results are consistent with the identification results of genetic sex of naked sturgeon obtained by dissection.

[0110] Example 14: Identification of genetic sex of Persian sturgeon

[0111] The steps of this example 14 can refer to the steps of example 1, and the difference is that after the genetic sex of Persian sturgeon is determined by dissection, the fin rays of 12 female Persian sturgeons and 12 male Persian sturgeons are taken, wherein the Persian sturgeons come from the sturgeon breeding farm in Xinjiang, China.

[0112] As shown in Figure 14, it can be known that 12 female Persian sturgeons appear two bands, and the sizes are 473bp and 717bp respectively; 12 male Persian sturgeons appear one band, and the size is 717bp. The experimental results are consistent with the identification results of genetic sex of Persian sturgeon obtained by dissection.

[0113] Example 15: Identification of genetic sex of high head sturgeon

[0114] The steps of this example 15 can refer to the steps of example 1, and the difference is that after the genetic sex of high head sturgeon is determined by dissection, the fin rays of 12 female high head sturgeons and 12 male high head sturgeons are taken, wherein the high head sturgeons come from the sturgeon breeding farm in Xinjiang, China.

[0115] As shown in Figure 15, it can be known that 12 female high head sturgeons appear two bands, and the sizes are 473bp and 717bp respectively; 12 male high head sturgeons appear one band, and the size is 717bp. The experimental results are consistent with the identification results of genetic sex of high head sturgeon obtained by dissection.

[0116] Example 16: Identification of genetic sex of middle snout sturgeon

[0117] The present embodiment 16 can refer to the steps of the embodiment 1, and the difference is that after the genetic sex of the Chinese paddlefish is determined by dissection, the fin rays of 12 female Chinese paddlefish and 12 male Chinese paddlefish are taken, wherein the Chinese paddlefish comes from the Chinese Xinjiang sturgeon farm.

[0118] As shown in Figure 16, it can be known that 12 female Chinese paddlefish all appear two bands, and the sizes are 473 bp and 717 bp respectively; 12 male Chinese paddlefish all appear one band, and the size is 717 bp. The experimental results this time are consistent with the genetic sex identification results of Chinese paddlefish obtained by dissection.

[0119] In summary, the present application is suitable for various sturgeons and has good universality. The genetic sex of the sturgeon identified by the present application is consistent with the genetic sex of the sturgeon obtained by dissection, spawning and squeezing sperm method or endoscopic technology, which shows that the present application can correctly identify the genetic sex of the sturgeon; compared with the dissection method, the present application only needs to extract trace amount of DNA of the sturgeon to be detected, and will not cause significant impact and damage to the sturgeon; compared with the spawning and squeezing sperm method and the endoscopic technology, the present application is not limited by the age of the sturgeon, and the genetic sex of the sturgeon can be identified throughout the life cycle. Since the primer combination of the present application provides two pairs of primers, the present application can also avoid experimental errors caused by unsuccessful DNA extraction, wrong or missing addition of drugs, and ensure the accuracy of sturgeon sex identification; and since the two DNA fragments amplified by the two pairs of primers are located on the same chromosome and the distance is not more than 20 kbp, the experimental error caused by serious DNA degradation can be effectively reduced. The present application can provide help for sturgeon sex control breeding and utilization of germplasm resources, and provide technical support for biodiversity research and Yangtze River protection.

[0120] Finally, it should be pointed out that the above embodiments are only used to illustrate the technical solutions of the present application, but not to limit them; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or make equivalent replacement for part or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A primer combination for identifying the genetic sex of sturgeon, wherein, comprises a first primer pair and a second primer pair; the first primer pair comprises a first primer and a second primer, the nucleotide sequence of the first primer is shown as SEQ ID NO: 1, and the nucleotide sequence of the second primer is shown as SEQ ID NO: 2; the second primer pair comprises a third primer and a fourth primer, the nucleotide sequence of the third primer is shown as SEQ ID NO: 3, and the nucleotide sequence of the fourth primer is shown as SEQ ID NO:

4.

2. The primer combination according to claim 1, wherein, The first primer pair is used for amplifying a female sturgeon specific DNA fragment, the nucleotide sequence of the female sturgeon specific DNA fragment is shown as SEQ ID NO: 5, and the fragment size is 473 bp; the second primer pair is used for amplifying a female sturgeon and male sturgeon common DNA fragment, the nucleotide sequence of the female sturgeon and male sturgeon common DNA fragment is shown as SEQ ID NO: 6, and the fragment size is 717 bp.

3. The primer combination according to claim 1 or 2, wherein, The sturgeon comprises at least one of Chinese sturgeon, Yangtze sturgeon, Small body sturgeon, Schizothorax chengtiangi, Large hybrid sturgeon, Siberian hybrid sturgeon, Siberian sturgeon, Huso dauricus, Russian sturgeon, Acipenser ruthenus, Acipenser sturio, Acipenser schrenckii, Acipenser persicus, Acipenser oxyrhynchus, Acipenser sturio.

4. A kit for genetic sex identification of sturgeon, wherein, The kit further comprises at least one of water, PCR buffer, dNTPs, DNA polymerase, and loading buffer.

5. The kit of claim 4, wherein, 6. Use of the primer combination of any one of claims 1-3 or the kit of any one of claims 4-5 in identifying the genetic sex of a sturgeon. comprises the following steps:

7. A method for identifying the genetic sex of a sturgeon, wherein, extracting DNA of a sturgeon sample to be detected; using the primer combination of any one of claims 1-3 or the kit of any one of claims 4-5 to perform PCR amplification on the DNA of the sturgeon sample to be detected as a template, to obtain a PCR amplification product; performing agarose gel electrophoresis detection on the PCR amplification product, when two bands of 473 bp and 717 bp appear in the detection result, it is identified that the genetic sex of the sturgeon is female; when one band of 717 bp appears in the detection result, it is identified that the genetic sex of the sturgeon is male. The sample of the sturgeon to be detected comprises at least one of fin bar and mucus of the sturgeon.

8. The method of claim 7, wherein, In the PCR amplification, the PCR reaction system comprises 3-5 μL of DNA template, 0.5-1.5 μL of the first primer, 0.5-1.5 μL of the second primer, 0.25-0.75 μL of the third primer, 0.25-0.75 μL of the fourth primer, 10-15 μL of 2×Taq Master Mix, and 4-6 μL of ddH2O.

9. The method of claim 7 or 8, wherein, In the PCR amplification, the PCR amplification program comprises 93-95℃ pre-denaturation for 3 min; 93-95℃ denaturation for 10 s, 54-56℃ annealing for 10 s, 72℃ extension for 19 s, for a total of 35 cycles; and finally 72℃ extension for 10 min.

10. The method according to any one of claims 7-9, wherein, ​

Images